Project Summary/Abstract Lymphocytes that stably reside in peripheral tissues play a critical role in defending against foreign pathogens, suppressing tumorigenesis and facilitating organ repair. Many of these cells first establish themselves in peripheral tissues during ?critical windows? of early postnatal development and are maintained throughout life within specific microanatomic niches, which provide the necessary factors for their survival and function. Understanding how tissue resident lymphocytes are established and maintained is of fundamental importance in our attempts to functionally manipulate these cells for therapeutic benefit. Abnormalities in tissue-resident lymphocytes and/or their tissue niches are thought to contribute to autoimmune and allergic inflammation. We have discovered that regulatory T cells (Tregs) in neonatal skin play a major role in suppressing the formation of a niche for T helper type 2 (Th2) cells in this tissue. Transient loss of Tregs in neonatal mice results in the aberrant outgrowth of a novel subset of stromal cells that preferentially express genes involved in type 2 immune responses (termed 'type 2 stromal cells' or `type 2 SCs'). Concurrently, a population of Th2 cells establishes residence in the same region of skin as type 2 SCs and persists into adulthood, long after complete restoration of the Treg compartment. We hypothesize that neonatal Treg dysfunction early in life predisposes to aberrant Th2 immune responses in adulthood by facilitating the establishment of a pathogenic Th2 cell niche in skin. Humans with congenital loss of Tregs have dysregulated Th2 immune responses in skin and develop severe atopic dermatitis (AD). Thus, we further postulate that Treg dysfunction early in life contributes to the development of human AD and that augmenting Tregs in AD skin will preferentially suppress Th2 immunity. Experiments outlined in this proposal will comprehensively define the Th2 niche in skin and determine the consequences of this niche for lifelong susceptibility to atopic inflammation. We will first mechanistically test whether type 2 SCs support Th2 cells and determine the molecular factors necessary for Th2 cell accumulation and maintenance in the skin. We will then test whether the establishment of the Th2 niche in neonatal life predisposes to atopic inflammation and aberrant AD-associated antimicrobial immune responses in adulthood. Finally, we will determine whether our findings translate to patients with AD. We will elucidate whether Tregs are defective in skin of pediatric patients with AD, whether a type 2 SC-Th2 cell axis exists in these patients, and whether Treg augmentation can suppress Th2 immune responses in AD skin. We have discovered a new stromal cell population in skin and have the tools to genetically manipulate these cells, allowing us to test a novel hypothesis centered around 'tissue imprinting' of immune cell niches early in life. We will utilize highly innovative technology to quantify T cell responses to cutaneous microbes. In addition, we will employ a novel ex vivo functional assay with human skin and a newly generated protein that selectively and potently activates Tregs. Thus, the experiments outlined in this proposal represent an innovative, comprehensive, and multidisciplinary approach to elucidate the cellular and molecular mechanisms that underpin cutaneous type 2 immune cell niches in both mouse models and in human disease.